1. Field of the Invention
The present invention relates to a process for producing non-proteinogenic L-amino acids by direct fermentation of microorganisms, and to L-amino acids obtained by the process.
2. The Prior Art
Non-proteinogenic amino acids are amino acids which are not used in nature as building blocks for protein biosynthesis and as a result may be clearly differentiated from the 20 proteinogenic amino acids. They are preferably xcex2-substituted L-alanine derivatives.
Non-proteinogenic amino acids are compounds of interest, for example, for the preparation of pharmaceuticals and agricultural active compounds. They can, as active compound or as a part of an active compound imitate, in a type of molecular mimicry, the structure of natural amino acids and as a result, for example, in receptor interactions cause a modulation of the natural reaction. In addition, they can serve quite generally as synthesis building blocks as chiral compounds in the context of the xe2x80x9cchiral poolxe2x80x9d.
Previous production processes for non-proteinogenic amino acids in enantiomerically pure form are generally based on complex syntheses which generally only permit access to a defined compound. Only a few processes enable different compounds to be produced by simple replacement of a starting material.
In most cases chemical syntheses are involved which themselves mostly start from the beginning from chiral building blocks or are followed by a racemate resolution.
Alternatively, some enzymatic processes are described. Thus, using transaminases, various non-proteinogenic amino acids can be prepared from xcex1-keto acids using L-glutamic acid as amino donor. A different process utilizes hydantoinases in combination with carbamoylases. However, enzymatic processes are also cost-intensive, since the corresponding enzymes must be provided and these have only a limited life as catalysts (Rehm et al., Biotechnology 1996; Vol. 6, pp. 505-560).
In contrast, processes for producing non-proteinogenic amino acids by direct fermentation of microorganisms would be particularly simple and expedient. However, such processes have the risk that the non-proteinogenic amino acid produced interferes with the metabolism of the natural amino acids and thus growth inhibition occurs. Previously, within this subject area, a process for the direct fermentation of D-amino acids has been disclosed (WO98/14602). This application describes the production of D-amino acids by recombinant microorganisms into which a D-amino transferase gene and an L-amino deaminase gene have been introduced. Furthermore, Saito et al. (Biol. Pharm. Bull. 1997, 20: 47-53) described the production of the plant non-proteinogenic amino acid L-pyrazolylalanine by expressing plant genes in Escherichia coli. The yields, however, are too low for commercial production, at  less than 1 g/l, and the costs, with the described use of L-serine as starting material, are very high.
It is an object of the present invention to provide an efficient process for producing a series of non-proteinogenic L-amino acids by direct fermentation.
This object is achieved according to the invention by a microorganism strain known per se having a deregulated cysteine metabolism being fermented in a manner known per se which comprises, during the fermentation, adding a nucleophilic compound to the fermentation batch in amounts such that this leads to the production of non-proteinogenic L-amino acids by the microorganism strain.
Preferably, at the end of the fermentation, the non-proteinogenic L-amino acids are separated off from the respective fermentation batch by means of methods known per se.
Surprisingly, it has been found that in the fermentation of microorganism strains having deregulated cysteine metabolism, instead of sulfide, a series of other nucleophilic compounds enter very efficiently into amino acid metabolism and the corresponding reaction products are excreted into the culture medium. Advantageously, glucose can be used here as an inexpensive source of carbon.
By means of the inventive addition of nucleophilic compounds during the fermentation, non-proteinogenic L-amino acids are accordingly formed. Preferably, therefore, a nucleophilic compound which enters into amino acid metabolism is added during the fermentation.
Preferably, nucleophilic compounds are added which comprise a radical selected from the group consisting of 
Particularly preferably, a nucleophilic compound selected from the following group is added to the fermentation batch:
Thiol of the general formula (1):
Hxe2x80x94Sxe2x80x94R1xe2x80x83xe2x80x83(1)
where R1 is monovalent substituted or unsubstituted alkyl, alkoxy, aryl or heteroaryl radical having a maximum of 15 carbon atoms;
azole of the general formula (2) or (3): 
xe2x80x83and their esters, ethers or salts,
where X and Y are identical or different and denote CR4 or N, and R4 is xe2x80x94H, xe2x80x94COOH, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NO2, xe2x80x94SH, xe2x80x94SO3, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, C1-C5-alkylcarbonyl or R1, and R1 has the meaning specified under formula (1) and
where R2 and R3 are identical or different and are R4 or where C1 and C2 in formula (3), instead of the substituents R2 and R3, are linked by means of a bridge [xe2x80x94CR5R6xe2x80x94]a, where a is 1, 2, 3 or 4, to form a ring, where R5 and R6 are identical or different and are R4 and one or more non-adjacent groups [xe2x80x94CR5R6xe2x80x94] can be replaced by oxygen, sulfur, or an imino radical, which may be unsubstituted or substituted by C1-C5-alkyl, and two adjacent groups [xe2x80x94CR5R6xe2x80x94] can be replaced by a group [xe2x80x94CR5xe2x95x90CR6xe2x80x94] or by a group [xe2x80x94CR5xe2x95x90Nxe2x80x94].
Isoxazolinone of the general formula (4) or (5): 
xe2x80x83and their esters, ethers or salts,
where X, R1, R2, R3 have the meaning specified above and where C1 and C2 in formula (5), instead of the substituents R2 and R3, can be linked by means of a bridge defined as for formula (3) to form a ring.
Examples of thiols are compounds selected from the group consisting of 2-mercaptoethanol, 3-mercaptopropanol, 3-mercaptopropionic acid, 3-mercapto-1-propanesulfonic acid, mercaptoethanesulfonic acid, 2-mercaptoethylamine, thioglycollc acid, thiolactic acid, thioacetic acid, mercaptosuccinic acid, mercaptopyruvic acid, dithiothreitol, dithioerythritol, 1-thioglycerol, thiophenol, 4-fluorothiophenol, 4-mercaptophenol, p-thiocresol, 5-thio-2-nitrobenzoic acid, 2-mercaptothiazole, 2-mercaptothiazoline, 2-mercaptoimidazole, 3-mercapto-1,2,4,-triazole, 2-thiophenethiol, 2-mercaptopyridine, 2-mercaptopyrimidine, 2-thiocytosine, 2-mercaptonicotinic acid, 2-mercapto-1-methylimidazole, 2-mercaptobenzothiazole, 2-mercaptobenzoxazole, 6-mercaptopurine.
Examples of azoles are compounds selected from the group consisting of 1,2-pyrazole, 3-methylpyrazole, 4-methylpyrazole, 3,5-dimethylpyrazole, 3-aminopyrazole, 4-aminopyrazole, pyrazole-4-carboxylic acid, pyrazole-3,5-dicarboxylic acid, 1,2,3-triazole, 1,2,4-triazole, 3-amino-1,2,4-triazole, 1,2,3,4-tetrazole, indazole, indazole-3-carboxylic acid, indazole-5-carboxylic acid, 5-aminoindazole, benzotriazole, benzotriazole-5-carboxylic acid, 5-aminobenzotriazole, aminopyrazolopyrimidine, 8-azaguanine, 8-azaadenine.
Examples of isoxazolinones are compounds selected from the group consisting of isoxazolin-2-one, 4-methylisoxazolin-2-one, 5-methylisoxazolin-2-one, 4,5-dimethylisoxazolin-2-one, 1,2,4-oxadiazolidin-3,5-dione.
Microorganism strains having deregulated cysteine metabolism that can be used in the inventive process are known from the prior art. They are distinguished by endogenic production of O-acetyl-L-serine, the immediate biosynthetic precursor of L-cysteine, which is increased in comparison with the wild type-strain. In a microorganism, it is known that in the last step of cysteine biosynthesis, due to the activity of O-acetyl-serine-sulfhydrylases, the acetyl function of the O-acetyl-L-serine is replaced by a thiol function and L-cysteine is thus formed. This reaction type is termed xcex2-substitution, since at the xcex2-carbon atom of the amino acid, a functional group is replaced.
Preferably, one of the following microorganism strains is used in the inventive process:
strains having modified cysE alleles, for example as described in WO 97/15673 (hereby incorporated by reference) or Nakamori S. et al., 1998, Appl. Env.
Microbiol. 64: 1607-1611 (hereby incorporated by reference) or Takagi H. et al., 1999, FEBS Lett. 452: 323-327, or
strains which contain efflux genes, as described, for example, in EP 0885962 A1 (equivalent to the U.S. application having the Ser. No. 09/097759 (hereby incorporated by reference)), or
strains having a modified CysB activity, as described in German patent application DE 19949579, or
strains which have been produced using nonspecific mutagenesis methods combined with screening methods for cysteine overproduction or reduced cysteine degradation, as described, for example, in WO 97/15673, or in Nakamori S. et al., 1998, Appl. Env. Microbiol. 64: 1607-1611.
Such strains are distinguished by the fact that, under adequate supply of an inorganic sulfur source, for example sulfate or thiosulfate, they excrete significant amounts of L-cysteine or a derivative thereof into the culture medium. As a result of the inventive addition of a nucleophilic compound during the fermentation, this compound enters into the xcex2-substitution and leads as a result to the production of non-proteinogenic L-amino acids.
With microorganism strains which do not have deregulated cysteine metabolism (for example the customary wild type organisms), such a procedure leads to slowing of cysteine biosynthesis and thus to growth inhibition. Therefore, no non-proteinogenic amino acids are formed in significant amounts.
Since the inventively used strains, however, have deregulated cysteine metabolism and thus a high endogenous level of O-acetyl-L-serine, it is possible to produce the non-proteinogenic L-amino acid in large amounts. At the same time, sufficient formation of L-cysteine is still ensured in order to guarantee cell growth of the microorganism.
Microorganism strains preferably used are those of the species Escherichia coli that have deregulated cysteine metabolism.
Preferably, these are Escherichia coli strains as described, for example, in WO 97/15673 or in EP 0885962 A1 (equivalent to the U.S. application having the Ser. No. 09/097759) or in DE 19949579. According to the processes described in these patent applications, cysteine metabolism can be deregulated in any strains by transformation with a plasmid that carries, for example, a feedback-resistant cysE allele and/or an efflux gene.
The inventive process for producing the non-proteinogenic L-amino acids using a microorganism strain is carried out in a fermenter in a manner known per se, but with additional addition of a nucleophilic compound.
The microorganism strain is grown in the fermenter as a continuous culture, as batch culture or, preferably, as fed-batch culture. Particularly preferably, a carbon source and a nucleophilic compound are continuously added during the fermentation.
Addition of the nucleophilic compound begins after inoculation, or, preferably, after an initial growth phase. Particularly preferably, the addition begins 6-8 hours after the start of fermentation and lasts until the end of fermentation.
The amount of added nucleophilic compound depends on its toxicity for the microorganism and is in the range from 10 to 1 000 mmol per liter of initial volume of the fermentation medium. Particular preference is given to an addition of 50 to 500 mmol per liter of initial volume of the fermentation medium.
Carbon sources for the fermentation are preferably sugars, sugar alcohols or organic acids. Particularly preferably in the inventive process, the carbon sources used are glucose, lactose or glycerol.
Preferably, glucose is added in a form which ensures that the content in the fermenter is kept in a range of 0.1-50 g/l during the fermentation. Particular preference is given to a range of 0.5-10 g/l.
The nitrogen source used in the inventive process is preferably ammonia, ammonium salts, or protein hydrolysates.
Further media additives which can be added are salts of the elements phosphorus, sulfur, chlorine, sodium, magnesium, nitrogen, potassium, calcium, iron and, in traces, (that is to say in xcexcM concentrations), salts of the elements molybdenum, boron, cobalt, manganese, zinc and nickel.
In addition, organic acids (for example acetate, citrate), amino acids (for example isoleucine), and vitamins (e.g. B1, B6) can be added to the medium.
Complex nutrient sources which can be used are, for example, yeast extract, corn steep liquor, soybean flour or malt extract.
The pH of the fermentation medium is in the range of 4-10. Preference is given to a range of 6-8. Particular preference is given to a pH range from 6.5 to 7.5.
The incubation temperature is 15-45xc2x0 C. Preference is given to a temperature of 30-37xc2x0 C.
The fermentation is preferably carried out under aerobic growth conditions. The oxygen is introduced into the fermenter using compressed air or pure oxygen.
Microorganisms which are fermented according to the described process excrete, in a fermentation time of 1 to 4 days, the corresponding non-proteinogenic L-amino acids into the culture medium with high efficiency.
When a nucleophilic substance is fed, microorganisms having deregulated cysteine metabolism excrete, during the fermentation, non-proteinogenic amino acids of the general formula (6) in the L configuration: 
where Z is a monovalent radical selected from the formulae (7) to (13) 
xe2x80x83and their esters, ethers or salts,
and R1, R2, R3, R4, X and Y have the meaning already specified under the formulae (1) to (5).
The inventive process makes it possible for the first time to produce compounds of the group 1,2,3,4-tetrazolyl-L-alanine and its derivatives, and 1,2,3-triazolyl-L-alanine and its derivatives. Preferably these are respectively the isomeric forms 1,2,3,4-tetrazol-1-yl-L-alanine (14) and 1,2,3,4-tetrazol-2-yl-L-alanine (15) and their derivatives including their esters, ethers or salts, and 1,2,3-triazol-1-yl-L-alanine (16) and 1,2,3-triazol-2-yl-L-alanine (17) and their derivatives, including their esters, ethers or salts, 
where R1, R2, R3 and R4 have the meaning specified above under the formulae (1) to (5).
The inventive process also makes it possible to produce for the first time compounds of the group S-heteroaryl-L-cysteines. These are in each case amino acid compounds having free amino and/or carboxylic acid functionalities. S-heteroaryl-L-cysteines are taken to mean cysteine derivatives which are characterized by substitution of a radical R7 of the S atom. Here, R7 is a heteroaryl radical that has aromatic character, is mono- or bicyclic, and, in addition to carbon atoms, has at least one heteroatom in a ring. Examples of heteroatoms are nitrogen, oxygen or sulfur. Heteroaryl radical can itself be substituted by a radical R4, where R4 has the meaning specified under formula (2). 
Examples of heteroaryl radicals are pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thienyl, thiazolyl, oxazolyl, furanyl, pyridyl, pyrimidyl, pyrazinyl, benzimidazolyl, benzotriazolyl, benzoxazolyl, benzothiazolyl or purinyl.
The invention therefore relates to said compounds.
Particularly preferred compounds are:
1,2,3,4-tetrazol-1-yl-L-alanine (R4=H)
1,2,3,4-tetrazol-2-yl-L-alanine (R4=H),
1,2,3-benzotriazol-1-yl-L-alanine (R2 and R3 are identical and are [xe2x80x94CR5xe2x95x90CR6xe2x80x94], where R5 and R6 are H and R2 and R3 are linked to form an aromatic ring),
1,2,3-benzotriazol-2-yl-L-alanine (R2 and R3 are identical and are [xe2x80x94CR5xe2x95x90CR6xe2x80x94], where R5 and R6 are H and R2 and R3 are linked to form an aromatic ring),
5-carboxy-1,2,3-benzotriazol-1-yl-L-alanine (R2 and R3 are different and are [xe2x80x94CR5xe2x95x90CR6xe2x80x94], where R5 and R6, in the case of R3, are H and, in the case of R2, R5 is H and R6 isxe2x80x94xe2x80x94COOH, and R2 and R3 are linked to form an aromatic ring),
5-carboxy-1,2,3-benzotriazol-2-yl-L-alanine (R2 and R3 are different and are [xe2x80x94CR5xe2x95x90CR6xe2x80x94], where R5 and R6, in the case of R3, are H and, in the case of R2, R5 is H and R6 is xe2x80x94COOH, and R2 and R3 are linked to form an aromatic ring),
1,2,4-triazol-3-yl-L-cysteine,
thiazol-2-yl-L-cysteine,
imidazol-2-yl-L-cysteine,
thien-2-yl-L-cysteine,
pyridin-2-yl-L-cysteine,
pyrimidin-2-yl-L-cysteine,
benzothiazol-2-yl-L-cysteine,
benzoxazol-2-yl-L-cysteine.
Preferably, the product, after separating off the biomass, is isolated from the culture supernatant by known methods (for example filtration, centrifugation). Such methods for isolating amino acids are also known to those skilled in the art. They comprise, for example, extraction, adsorption, ion-exchange chromatography, precipitation, crystallization.
The examples below serve for further explanation of the invention. The bacteria strain Escherichia coli W3110/pA-CYC184-cysEX-GAPDH-ORF306, which was used for carrying out the examples, was deposited at the DSMZ (Deutsche Sammlung fxc3xcr Mikroorganismen und Zellkulturen GmbH, D-38142 Braunschweig) under the number DSM 13495 in accordance with the Budapest Treaty.